Over the last two decades, the use of DNA barcodes has transformed our ability to identify and assess life on our planet. Both strengths and weaknesses of the method have been exemplified through thousands of peer-reviewed scientific articles. Given the novel sequencing approaches, currently capable of generating millions of reads at low cost, we reflect on the questions: What will the future bring for DNA barcoding? Will identification of species using short, standardized fragments of DNA stand the test of time? We present reflected opinions of early career biodiversity researchers in the form of a SWOT analysis and discuss answers to these questions.

Environmental DNA (eDNA) metabarcoding is revolutionising the study of aquatic ecosystems, enabling high-throughput analysis of biodiversity with minimal disturbance. Despite its potential to support fisheries management, species identification and downstream analysis reliability are hindered by the lack of standardisation in DNA fragment choice. This study compares the species discrimination power of three markers used in marine fish (e)DNA (meta)barcoding – 12S rRNA, 16S rRNA and cytochrome oxidase subunit I (COI) – as well as two amplicons for each. We analysed sequences from NCBI GenBank for 10 orders of Actinopterygii (ray-finned fishes), including mitochondrial genomes. We assessed species discrimination by determining the percentage of monophyletic species in Neighbour-Joining trees and calculating congeneric divergences for two datasets: one with genomic regions extracted from mitochondrial genomes (771 species) and another with independent sequences for each region (3,879 species). Amongst (meta)barcoding amplicons in the mitochondrial genomes’ dataset, the COI Folmer and Leray-Lobo regions had the highest discriminatory power, with 89.2% and 87.0% monophyletic species, respectively, while the 12S Teleo region had the lowest at 71.6%. Conversely, using independent sequences of these amplicons, Folmer and Leray-Lobo had the lowest percentages of monophyletic species, at 64.8% and 63.5%, respectively, while Actinopterygii 16S (Ac16S) had the highest at 83.0%. Species discrimination is influenced by the marker’s evolutionary rate, fragment length, target fish order and the quality of reference sequence data. We recommend considering species discriminatory power differences for amplicon selection, especially for species-level identifications. We advise a standard multi-marker approach under certain scenarios, particularly when the presence of close congeneric species is expected.

Biodiversity studies greatly benefit from molecular tools, such as DNA metabarcoding, which provides an effective identification tool in biomonitoring and conservation programmes. The accuracy of species-level assignment, and consequent taxonomic coverage, relies on comprehensive DNA barcode reference libraries. The role of these libraries is to support species identification, but accidental errors in the generation of the barcodes may compromise their accuracy. Here we present an R-based application, BAGS (Barcode, Audit & Grade System), that performs automated auditing and annotation of cytochrome c oxidase subunit I (COI) sequences libraries, for a given taxonomic group of animals, available in the Barcode of Life Data System (BOLD). This is followed by implementing a qualitative ranking system that assigns one of five grades (A to E) to each species in the reference library, according to the attributes of the data and congruency of species names with sequences clustered in Barcode Index Numbers (BINs). Our ultimate goal is to allow researchers to obtain the most useful and reliable data, highlighting and segregating records according to their congruency. Different tests were performed to perceive its usefulness and limitations. BAGS fulfils a significant gap in the current landscape of DNA barcoding research tools by quickly screening reference libraries to gauge the congruence status of data and facilitate the triage of ambiguous data for posterior review. Thereby, BAGS have the potential to become a valuable addition in forthcoming DNA metabarcoding studies, in the long term contributing to globally improve the quality and reliability of the public reference libraries.

The John Dory Zeus faber is a commercially exploited demersal fish species with a known distribution ranging from the Northeast Atlantic to parts of the Indian and Pacific oceans. A previous genetic survey using cytochrome c oxidase subunit I (COI) DNA barcodes suggested the presence of two geographically segregated taxonomic units within Z. faber . We revisit this hypothesis by expanding the number and geographic coverage of DNA barcodes, addressing a major data gap along parts of the Atlantic coast of Africa and conducting a comprehensive phylogeographic analysis. Our findings consolidated the existence of two highly divergent mitochondrial clades, Clade A and Clade B (mean K2P distance: 7.4%), with the transition zone between them located along the Atlantic coast of Morocco. Clade A exhibited no phylogeographic structure, with haplotypes shared between Northeast Atlantic and Mediterranean populations. Conversely, four geographically structured subclades (mean K2P distance: 0.9%) were detected within Clade B, extending south and eastward from Morocco to Japan and New Zealand. Historical demographic events driving allopatric divergence, along with oceanographic and environmental factors, likely shaped the current geographic distribution of the two clades. These findings not only prompt the need to re‐evaluate the taxonomic status of Z. faber but also highlight the probable existence of multiple evolutionarily significant units (ESUs) that must be considered in the scope of stock assessment, fisheries management and conservation purposes.

Environmental DNA (eDNA) metabarcoding is revolutionizing the study of aquatic ecosystems, enabling high-throughput analysis of biodiversity with minimal disturbance. Despite its potential to support fisheries management, species identification and downstream analysis reliability are hindered by the lack of standardization in DNA fragment choice. This study compares the species discrimination power of three markers used in marine fish eDNA metabarcoding —12S rRNA, 16S rRNA, and cytochrome oxidase subunit I (COI)— as well as two amplicons for each. We analyzed sequences from NCBI GenBank for 10 orders of Actinopterygii, including mitochondrial genomes. We assessed species discrimination by determining the percentage of monophyletic species in Neighbor-Joining trees and calculating average congeneric divergences for two data sets: one with genomic regions extracted from mitochondrial genomes (771 species) and another with independent sequences for each region (3879 species). Among (meta)barcoding amplicons, for the mitochondrial genomes’ dataset, the Folmer and Leray-Lobo regions had the highest discriminatory power, with 89.2% and 87% monophyletic species, respectively, while the 12S Teleo region had the lowest at 71.6%. Conversely, using independent sequences, the Folmer and Leray-Lobo regions had the lowest percentages of monophyletic species, at 64.8% and 63.5%, respectively, while Ac16S had the highest at 83.0%. Species discrimination is influenced by the marker’s substitution rate, fragment length, target fish order, and the quality of reference sequence data. We recommend considering species discriminatory power differences for amplicon selection, especially for species-level identifications. We advise a standard multi-marker approach under certain scenarios, namely when the presence of close congeneric species is expected.